|
| Status |
Public on Oct 19, 2020 |
| Title |
colon_water_4 |
| Sample type |
RNA |
| |
|
| Source name |
colon mucosa, drinking water
|
| Organism |
Mus musculus |
| Characteristics |
strain: Balb/cJ
gender: female
tissue: colon mucosa
model_induction: water
compound: vehicle control
|
| Treatment protocol |
Chronic colitis was induced in eight weeks old mice by cyclical administration of 4% DSS via drinking water, involving 3 cycles of DSS for 4 days interrupted by washout periods of 3 days under regular drinking water. The mice (n=5 per group) were assigned in 3 groups: 1/ Drinking water, 2/ DSS in water and filgotinib vehicle 3/ DSS in water and filgotinib. Filgotinib was given at 30 mg/kg, once a day by oral gavage.
|
| Growth protocol |
Standard animal housing conditions
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA preparation from mouse colon, mucosal samples were obtained by cutting lengthwise thirty millimeters of the most distal part of descending colon and scraping mucosa from the connective tissue. Mucosa was sampled into homogenization tubes containing 1.4 mm ceramic beads and was immediately snap frozen in liquid nitrogen. Mucosa samples were homogenized using the Precellys 24 homogenizer (Bertin Technologies™, France). Total RNA were isolated and further purified with NucleoSpin 96 RNA Tissue Core Kit (Macherey-Nagel™) according to the manufacturer’s instructions. RNA yield were determined by spectrophotometry at 260/280 nm.
|
| Label |
Cy3
|
| Label protocol |
Before labeling reaction, samples are diluted to 100 ng/ml. Then, Agilent Technologies’ Low Input Quick Amp Labeling Kit generates fluorescent cRNA with 100 ng of total RNA. The method first uses reverse transcriptase to synthesize the first and second strands of cDNA and then T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3-labeled CTP. Before proceeding to the sample hybridization, labeled cRNA and dye concentrations are quantified with NanoDrop ND-1000 spectrophotometer (Thermo Scientific): - cRNA concentration (ng/μl), - Cyanine 3 concentration (pmol/μl). After labeling step, cRNA sample size ranges from 50 to 3000 nucleotides. Thus, fragmentation is required to take away secondary structures. Fragmentation step is achieved with specific buffer (Agilent Technologies) and allows obtaining cRNA length between 50 to 200 nucleotides and then optimal hybridization with Agilent 60-mer oligonucleotide microarrays. This step is incubation at 60°C for 30 minutes.
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| |
|
| Hybridization protocol |
600 ng of Cyanine 3-labeled amplified cRNA are hybridized at 65°C for 17 hours.
|
| Scan protocol |
Scanning is performed with Agilent Technologies’ scanner using default parameters for 8x60k format (61*21.6 mm; 3 micrometer scan resolution; 20 bit TIFF; green channel)
|
| Description |
Gene expression of control animals (drinking water)
|
| Data processing |
Data are extracted with Feature Extraction 11.5.1.1 software (Agilent Technologies). This software reads and processes microarray image files to prepare them for analysis. Feature Extraction automatically assigns a grid template and a protocol, based on the barcode of the slide. It determines feature intensities, rejects outliers and calculates statistical confidences. Quality was assessed using the arrayQualityMetrics R/BioConductor package. A filter was set to remove probes that are not expressed above background (cut-off defined such that a probe must have a valid and above background signal in at least 40% of the samples). Background correction (using the “normexp” method with an offset of 16) and quantile normalization were applied to obtain log2 transformed and normalized expression data. Within-array replicate probes are summarized with the mean. Differential expression analysis was performed using empirical Bayes methods and generalized linear models (limma R/BioConductor package). All data is MIAME compliant
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| |
|
| Submission date |
Oct 20, 2017 |
| Last update date |
Oct 19, 2020 |
| Contact name |
Maté Ongenaert |
| E-mail(s) |
mate.ongenaert@glpg.com
|
| Phone |
+3215342927
|
| Organization name |
Galapagos
|
| Street address |
Generaal De Wittelaan L11A3
|
| City |
Mechelen |
| ZIP/Postal code |
2800 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL21163 |
| Series (1) |
| GSE105417 |
Selective inhibition of JAK1 with filgotinib reverses pathogenic processes in the DSS preclinical model for IBD |
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